Systematic monitoring of protein complex composition and abundance by blue-native PAGE.
Systematic monitoring of protein complex composition and abundance by blue-native PAGE.
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通过蓝色原生 PAGE 系统监测蛋白质复合物的组成和丰度。
DOI：
10.1101/pdb.prot5221
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发表时间：
2009
影响因子：
--
通讯作者：
A. Millar
中科院分区：
文献类型：
--
作者：
Holger Eubel;A. Millar
许多多肽在体内不作为单个自主单元执行其功能。相反，多个多肽缔合以形成更高分子量的结构。蓝色聚丙烯酰胺凝胶电泳（BN-PAGE）允许一系列的主要蛋白质复合物参与这样的蛋白质蛋白质相互作用，以同时可视化，并在一个单一的实验。当与十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）的第二维结合时，BN/SDS-PAGE程序可以根据复合物的分子量来解析复合物，以及根据亚基的分子量来解析每个复合物内的亚基。类似地，与差异凝胶电泳（DIGE）结合使用，它可以准确地定量不同样品之间或同一样品中不同复合物之间蛋白质复合物丰度或亚基组成的变化。以下基本方案描述了第一维度（BN-PAGE）和第二维度（SDS-PAGE）的样品制备和凝胶浇铸。提供了有和没有DIGE标记的变体，沿着荧光DIGE技术所需的额外步骤。
Many polypeptides do not perform their functions as single autonomous units in vivo. Instead, multiple polypeptides associate to form higher molecular mass structures. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) allows a range of the major protein complexes involved in such proteinprotein interactions to be visualized simultaneously and in a single experiment. When combined with a second dimension of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the BN/SDS-PAGE procedure can resolve the complexes according to their molecular weight, as well as the subunits within each complex, according to the molecular weights of the subunits. Similarly, used in conjunction with differential in-gel electrophoresis (DIGE), it can accurately quantify changes in protein complex abundance or subunit composition between different samples, or between different complexes within the same sample. The following basic protocol describes sample preparation and gel casting for the first (BN-PAGE) and second (SDS-PAGE) dimensions. Variants are presented with and without DIGE labeling, along with the additional steps required for the fluorescence DIGE technique.